For a virus to complete an infectious cycle, newly assembled particles must have a mechanism to escape the infected cell. Enveloped viruses accomplish this egress event through processes that include budding directly from the cell surface or secretion from the cell in a membrane-bound form. Non-enveloped viruses, however, face a challenge during egress: how to cross the plasma membrane of an infected cell during release. Traditionally, non-enveloped viruses were thought to disrupt cell membrane integrity to promote efficient egress, thereby leading to cell death. However, studies of several non-enveloped viruses, including parvovirus, poliovirus, and rotavirus, have challenged this theory, revealing that mature particles can exit infected cells without compromising cell viability, a phenomenon termed non-cytolytic egress. We have observed that reovirus, a non-enveloped enteric virus capable of hematogenous dissemination, can escape from infected human brain microvascular endothelial cells (HBMECs) without altering cell viability. There is limited understanding of how non-enveloped viruses, including reovirus, exit infected cells without causing cell death. I have proposed two integrated specific aims to fill knowledge gaps about the mechanisms underlying reovirus egress from mammalian cells. In Specific Aim 1, I will identify host genes required for non-cytolytic reovirus egress from HBMECs using RNAi screening. Candidate reovirus egress mediators will be validated using genetic, biochemical, and pharmacological approaches to elucidate mechanisms by which individual host factors promote reovirus exit. In Specific Aim 2, I will define the trajectory of reovirus particles during egress, image particle release from infected cells, and visualize the subcellular machinery associated with reovirus egress. Live-cell microscopy will be paired with correlative light and electron microscopy of reovirus-infected cells to define the cellular structures that colocalize with egressing reovirus particles. Collectively, this work will offer new insights into strategies used by non-enveloped viruses to escape infected cells and elucidate cellular pathways involved in transport of macromolecular cargo destined for the extracellular space.